Toll-like receptor agonists targeted to cancer cell metabolism

Abstract

Imidazoquinoline Toll-Like Receptor (TLR) agonists such as Imiquimod are effective immunotherapeutics in the treatment of basal skin cell carcinoma, and more recently, melanoma. However, direct injection of imidazoquinoline TLR agonists causes systemic inflammation and autoimmune side-effects thereby limiting clinical use to topical applications. Here we use a metabolic caging strategy to confine the immunostimulatory effects of imidazoquinoline TLR agonists to tumor microenvironment. We have covalently attached a galactopyranoside metabolic cage to the primary amino group, critical for activity, of the TLR7 agonist Imiquimod. Addition of the metabolic cage abrogates TLR agonism as measured by NFκB transcription in RAW-Blue cells or inflammatory cytokines produced by primary murine Bone Marrow Derived Dendritic Cells (BMDCS). The galactopyranoside cage is efficiently removed via enzymatic cleavage from exogenously added β-galactosidase resulting in activation of immune cells that is comparable to native agonist. Enzymatic cleavage also occurs using β-galactosidase that is overexpressed in B16 melanoma cells. This results in increased immunostimulatory effects in co-cultures of BMDCs and B16 melanoma cells relative to BMDCs co-cultured with healthy melanocytes. We anticipate this will provide a general framework upon which TLR agonists can be targeted to the tumor microenvironment.